PROJECT SUMMARY/ABSTRACT More than 30 years into the global HIV-1 epidemic, novel HIV-1 prevention strategies are still being sought. A challenge to the design of an effective HIV-1 vaccine is the lack of known natural correlates of protection from infection. A relevant model to identify such correlates is offered by individuals who remain seronegative despite repeated HIV-1 exposures (HIV-1-exposed seronegatives, HESN). Studies of HESN have uncovered evidence of potential immune correlates of protection from HIV-1 infection in the form of HIV-specific immune responses and reduced immune activation, or immune quiescence. However, the vast majority of these studies were focused on immunity within the peripheral blood rather than mucosal sites of initial virus entry and replication. Thus, we plan to use mucosal sampling of a novel cohort including male and female HESN from HIV- serodiscordant couples and HIV-unexposed (HUSN) men and women, combined with state-of-the-art immunological techniques, to identify novel mucosal immune mechanisms of protection from HIV-1. Given our recent findings of an association of increased cervicovaginal HIV-neutralizing IgA and higher HIV- 1 exposure, we hypothesize that immune responses within the genital mucosa will be altered in HESN compared to HUSN. Thus, in Aim 1 we will examine local T cell responses in the genital mucosa of HESN compared to HUSN, and we expect to observe increased responses and retention of these immune cells in individuals with higher HIV-1 exposure. As we and others have shown that immune quiescence appears to offer protection from HIV-1 infection, we propose to examine both HIV-specific immunity as well as immune activation in HESN and HUSN longitudinally. In parallel, we will examine the stability of these local T cell responses in the genital tract upon PrEP initiation by HESN men and women, as we have evidence of higher magnitude mucosal immune responses in PrEP users. Finally, using previously archived samples from the nested Partners PrEP case-control study, we will in test whether the novel changes in immune signatures we identify are correlated with protection from HIV-1 infection. In Aim 2, following up on our finding of differential cervicovaginal HIV-neutralizing IgA in women with higher HIV-1 exposure and with PrEP use, we will examine mechanisms of local antibody-secreting cells within the genital tract of men and women following HIV-1 exposure and PrEP initiation. In sum, upon completion of our proposed studies, we will have a complete characterization of adaptive immune responses and activation in the male and female genital tracts and blood in HESN upon HIV-1 exposure, with measures of the changes that occur longitudinally upon PrEP treatment. These studies will potentially inform vaccine and other HIV prevention strategies though identification of immune correlates of protection, and will also address a critical need to better understand mechanisms of the human mucosal immune responses, as such work will enhance our knowledge of the immunopathology of and immune-mediated protection to a range of chronic and infectious diseases.